In this study, 25 actinomyces isolates were obtained from 10 different poultry farms and tested for their keratinase activity. The isolate with the highest keratinase activity was identified through molecular identification by PCR and sequencing of the 16S rRNA gene to be Streptomyces spp. and was named Streptomyces werraensis KN23 with an accession number of OK086273 in the NCBI database. Sequential mutagenesis was then applied to this strain using UV, H2O2, and SA, resulting in several mutants. The best keratinolytic efficiency mutant was designated as SA-27 and exhibited a keratinase activity of 106.92 U/ml. To optimize the keratinase expression of mutant SA-27, the Response Surface Methodology was applied using different parameters such as incubation time, pH, carbon, and nitrogen sources. The optimized culture conditions resulted in a maximum keratinase specific activity of 129.60 U/ml. The genetic diversity of Streptomyces werraensis KN23 wild type compared with five mutants was studied using Inter-simple sequence repeat (ISSR). The highest total and polymorphic unique bands were revealed in the S. werraensis KN23 and SA-18 mutant, with 51 and 41 bands, respectively. The dendrogram based on combined molecular data grouped the Streptomyces werraensis and mutants into two clusters. Cluster I included SA-31 only, while cluster II contained two sub-clusters. Sub-cluster one included SA-27, and sub-cluster two included SA-26. The sub-cluster two divided into two sub-sub clusters. Sub-sub cluster one included SA-18, while sub-sub cluster two included one group (SA-14 and S. werraensis KN23).

No abstract

The most abundant organic carbon source on Earth is cellulosic materials. Its main resources are crop straws which are not commonly used and produce environmental pollution. These resources can be a site of biological hydrolysis to primary sugars by cellulase enzymes, in which avicelase is the most efficient enzyme in the cellulase family. This work aimed to clone the avicelase gene, transfer it to E. coli, optimize its expression, saccharify rice straw to its primary sugars, and ferment it to bioethanol. The avicelase gene was cloned from the Bacillus subtilis strain and cloned into two E. coli (i.e., DH5α and Bl21) strains. The optimized avicelase activity was described by testing the effect of different media and growth conditions including different carbon and nitrogen sources, as well as pHs and shaking or static conditions. Avicelase enzyme was extracted and used to saccharify rice straw. The obtained glucose was subjected to fermentation by Saccharomyces cerevisiae F.307 under an aerobic condition growth for the production of bioethanol. The ethanol yield was 5.26% (v/v), and the fermentation efficiency was 86%. This study showed the ability to clone one of the cellulolytic genes (i.e., avicelase) for the valorization of rice straw for producing renewable energy and bioethanol from cellulolytic wastes such as rice straw.